Re-examining class-I presentation and the DRiP hypothesis.

MHC class I molecules present peptides derived from intracellular proteins, enabling immune surveillance by CD8(+) T cells and the elimination of virus-infected and cancerous cells. It has been argued that the dominant source of MHC class I-presented peptides is through proteasomal degradation of newly synthesized defective proteins, termed defective ribosomal products (DRiPs). Here, we critically examine the DRiP hypothesis and discuss recent studies indicating that antigenic peptides are generated from the entire proteome and not just from failures in protein synthesis or folding.

Substrate-induced protein stabilization reveals a predominant contribution from mature proteins to peptides presented on MHC class I.

The origin of the MHC class I-presented peptides are thought to be primarily from newly synthesized but defective proteins, termed defective ribosomal products. Most of the data supporting this concept come from studies in which inhibitors of protein synthesis were found to rapidly block Ag presentation even when cells contained a pool of mature proteins. However, these data only indirectly address the origin of presented peptides, and in most studies, the contribution of mature functional proteins to the class I peptide pool has not been directly quantified. In this report, we address the efficiency and contribution of mature proteins using a tetracycline-inducible system to express Ags that are conditionally stabilized upon ligand binding. This system circumvents the use of general inhibitors of protein synthesis to control Ag expression. Moreover, by controlling Ag stabilization, we could investigate whether the degradation of mature Ags contributed to Ag presentation at early and/or late time points. We show that mature proteins are the major contributor of peptides presented on class I for two distinct antigenic constructs. Furthermore, our data show that the protein synthesis inhibitors used previously to test the contribution of defective proteins actually block Ag presentation in ways that are independent from blocking Ag synthesis. These data suggest that for the constructs we have analyzed, mature functional proteins, rather than defective ribosomal products, are the predominant source of MHC class I-presented peptides.

Using intein catalysis to probe the origin of major histocompatibility complex class I-presented peptides.

All vertebrate nucleated cells generate peptides from their expressed gene products and then display them at the cell surface bound to MHC class I molecules. This allows CD8(+) T cells to detect and eliminate abnormal cells that are synthesizing foreign proteins, e.g., from viruses or mutations. To permit the immune system to more uniformly monitor a cell's proteins, regardless of their half-life or location, it has been thought that the products of rapid degradation of the mistakes of protein synthesis (defective ribosomal products, DRiPs) preferentially contribute to the class I-presented peptides. However, using intein catalysis to generate peptide sequences exclusively by posttranslational splicing of mature proteins, we show here that presented peptides can be generated from fully folded and functional proteins. Remarkably, the presentation of peptides from two model mature proteins is just as efficient as from newly synthesized proteins subject to errors in translation or folding. These results indicate that for the constructs we have analyzed, DRiPs are not a more efficient source of class I peptides for antigen presentation than the turnover of mature functional proteins. Accordingly, our data suggest that one of the major ways the immune system evaluates the health of cells is by monitoring the breakdown products of the proteome.

Proteases in MHC class I presentation and cross-presentation.

Cells that have mutated their genes or are virally infected are a potential threat to a host. Consequently, the immune system has evolved mechanisms for CD8 T lymphocytes to identify such cells and eliminate them. The generation of CD8 T cell responses occurs in two phases, both of which critically involve the process of Ag presentation. In the first phase, sentinel cells gather Ags present in tissues and then present them to naive CD8 T cells in ways that stimulate their maturation into effectors. In the second phase, these effector cells seek out and eliminate the pathological cells. The abnormal cells are identified through their presentation of immunogenic Ags that they are producing. The Ag presentation mechanisms used by the sentinel cells can be different from those in other cells. This article will review these mechanisms with a focus in each case on how antigenic peptides are generated for presentation.

The dynamics of immunoglobulin V-gene usage and clonotype expansion in mice after prime and boost immunizations as analyzed by NGS.

In the current study, an improved NGS approach was developed to study the B-cell repertoire evolution in a simple mouse immunization model including only two DNA immunizations. The combination of 5'RACE and Ion Torrent long reads enabled unbiased immunoglobulin repertoire analysis even from small amounts of peripheral mouse blood. The B-cell population expanded by the vaccine displayed a relatively strong clonality. Upon priming with the first vaccine dose, we observed a consistent pattern of V-segment gene and CDR3 usage (public specificities). Interestingly, this pattern diversified with the second dose of immunization -it was relatively different in individual mice in spite of having received the same vaccine regimen (private specificities). Nevertheless, there were several instances in which the same public V-segment genes and CDR3s that were expanded after the first dose were further amplified after the second immunization. Taken together, it appears that the major clonotypes expanded by vaccination were originally a homogeneous subset that later diversified after a second dose leading to diverse "private" clonal compositions in different mice. These results established a new platform valuable to perform longitudinal analyses of the Ig germline gene usage and clonotype evolution throughout an immunization regimen in a commonly used animal model.

Relative contributions of codon usage, promoter efficiency and leader sequence to the antigen expression and immunogenicity of HIV-1 Env DNA vaccine.

Optimized antigen expression is critical to the immunogenicity of DNA vaccines. A number of approaches have been proposed to enhance the antigen expression and/or immunogenicity of DNA vaccines, but their relative contributions have not been compared in a same antigen system. In the current study, optimization of codon usage, enhancement of viral promoter function and selection of secretary leader sequences were evaluated for their roles in improving the immunogenicity of a same model antigen, the HIV-1 envelope glycoprotein. Our data demonstrated that all these factors can work synergistically to improve the final antigen expression and immunogenicity of HIV-1 Env DNA vaccines, indicating they work through different mechanisms. The best result came from the approach that optimized all three components in a DNA vaccine design. Our study further revealed that the levels of HIV-1 env-specific RNA transcripts in transiently transfected 293T cells were higher from the codon-optimized gene than the wild type counterpart. This finding suggested other mechanism may also contribute to the increased antigen expression and immunogenicity of codon-optimized DNA vaccines in addition to the improved tRNA usage in mammalian cells for codon-optimized viral genes as previously reported.